Identifier

Author

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Vibrio vulnificus is a Gram-negative organism that is found in estuarine environments. The bacterium is capable of causing gastroenteritis, wound infections, and septicemia, often resulting in death for susceptible individuals. Monoclonal antibodies (MAbs) to five lipopolysaccharide (LPS) O-serovars have previously been used as a serotyping system in epidemiological studies. Here is the first report of two additional MAbs capable of recognizing two new LPS serovars (O6 and O7), further increasing the number of V. vulnificus that can be typed. Analysis of V. vulnificus isolates from the United States, Europe, and Asia demonstrated that the O1/O5 serovar is dominate in clinical isolates, serovars O3 and O4 are dominate in the environmental isolates of Europe, and that most environmental isolates from United States are nontypeable. Additionally, MAb, RS1, was chosen for further analysis by having DNA coding for its light and heavy chains cloned into a phage-display system. The published primers, designed to be used with the pComb3X display system, produced primer dimers and were unsuccessful in generating the single chain variable fragment (scFv) to be cloned into the phage vector. Modification of the existing primers inhibited the formation of primer dimers and allowed for the successful generation of the scFv. The resulting phage-displayed antibody, PJ1, displayed similar binding characteristics as RS1 showing the usefulness of phage-display as an alternative method of maintaining pre-existing MAbs. Lastly, it was proposed that as V. vulnificus undergoes capsular switching it is possible that LPS switching occurs as well. Analysis of the data suggests that capsule and LPS switching are not directly related. Interestingly, LPS switching seems to occur in a unidirectional manner on two separate branches that converge on serovar O5 suggesting that O5 may be a group of sugars that are used as a building block for other serovars. This allowed for the formation of a speculative LPS model. The stability of serovars O6 and O7 in the LPS switching assay make them difficult to place in relationship to serovar O5 and further analysis will be needed to more accurately place them on the LPS model.